Previously published studies have demonstrated expression of FGF2 and FGFRs in human lung cancers and in NSCLC cell lines (Berger et al., 1999
; Chandler et al., 1999
; Kuhn et al., 2004
). Also, rare somatic mutations in FGFR1 that may confer gain-of-function have been identified (Zhao et al., 2005
) and amplification of the FGFR1 gene has been detected in human NSCLC, albeit at a very low frequency (Davies et al., 2005
). Our results support these previous studies and also provide molecular evidence for an active FGF autocrine signaling pathway in a subset of NSCLC cell lines. Our demonstration of an active FGF-FGFR autocrine loop in NSCLC cell lines also provides a mechanism for the observed insensitivity of some NSCLC tumors and cell lines to EGFR-specific TKIs. Our data suggest that gefitinib-insensitive NSCLC cell lines employ alternative receptor tyrosine kinases, such as the FGFR, to establish self-sufficiency in growth. Previous studies have shown that gefitinib-sensitive NSCLC tumors and cell lines are enriched for the adenocarcinoma and bronchoalveolar carcinoma histological type (Miller et al., 2004
). By contrast, among the NSCLC cell lines where we demonstrated FGFR-dependent autocrine signaling, H226, H520 and H1703 are derived from squamous cell carcinomas and H661 and H1299 are derived from large cell carcinomas. Thus, FGFR inhibitors may better target the squamous cell and large cell histologies of NSCLC cell lines that frequently exhibit a high degree of insensitivity to EGFR TKIs.
A recent study (Rikova et al., 2007
) employed a phosphotyrosine proteomic approach to survey for tyrosine kinases that are active in lung cancer cell lines and primary tumors. In contrast to our present findings, only 3 of 41 NSCLC cell lines showed evidence for activated FGFR1, suggesting that the FGF/FGFR pathway is not a major receptor pathway active in NSCLC cells lines. It is noteworthy that the NSCLC cell line panel employed in the aforementioned study was comprised of ~75% adenocarcinomas while we find that squamous cell and large cell carcinomas are enriched in the FGF and FGFR-expressing NSCLC cell lines. In addition, each experimental approach (proteomics, genomics, etc) will have unique biases and tyrosine-phosphorylated peptides derived from FGFR1 or FGFR2 may not be efficiently detected by the experimental approaches employed. Alternatively, the selected phosphopeptides from FGFR1 or FGFR2 may not serve as sensitive indicators of FGFR activity in cell lines or tumors.
A conclusion from our experiments is that FGFR1 IIIc and/or FGFR2 IIIc and their respective ligands, FGF2 and FGF9, undergo co-selection as components of an autocrine signaling pathway during initiation and progression of NSCLC. It is noteworthy that no NSCLC cell line expressing FGFR1 IIIc or FGFR2 IIIc was found that did not also express FGF2 or FGF9. However, HCC827 and HCC4006 expressed FGF2, but lacked detectable expression of FGFR1 IIIc or FGFR2 IIIc. As single markers, FGFR1 IIIc or FGFR2 IIIc may be a more reliable indicator of FGF and FGFR-dependent autocrine signaling. FGFR signaling is critical for lung development and tissue homeostasis, although FGFR2 IIIb, FGF7 and FGF10 are the key players in this regard (Eswarakumar et al., 2005
). However, our studies did not detect co-expression of FGFR2 IIIb with FGF7 or FGF10 in human NSCLC cell lines. Rather, FGFR1 IIIc and FGFR2 IIIc are co-expressed with their high-affinity ligands, FGF2 or FGF9. The literature indicates that FGF2 and FGFR1 appear to be most highly expressed in vascular compartments of the lung (Powell et al., 1998
). Thus, ectopic expression of FGF2, FGF9, FGFR1 IIIc and FGFR2 IIIc may be induced during lung tumorigenesis. In support of this possibility, enhanced bronchial expression of FGF2 and FGFR1 is observed in chronic obstructive pulmonary disease (Kranenburg et al., 2002
; Kranenburg et al., 2005
) where the risks for chronic obstructive pulmonary disease and lung cancer aggregate (Schwartz and Ruckdeschel, 2006
). An alternative mechanism for the observed co-expression of specific FGFs and FGFRs in NSCLC cell lines is that a specific lung epithelial cell type or progenitor may express FGF2 or FGF9 and FGFR1 IIIc and/or FGFR2 IIIc and serve as a precursor for the NSCLC cells that exhibit an FGFR autocrine signaling loop. In support, a recent study by Ince et al revealed a higher degree of gene expression similarity between a given non-transformed mammary epithelial cell type and its transformed derivative than between different types of non-transformed mammary epithelial cell precursors (Ince et al., 2007
), indicating that tumor cells retain a high degree of gene expression similarity with their non-transformed precursors.
In addition to autocrine signaling through EGFR and FGFR pathways in NSCLC, the literature documents that multiple receptor tyrosine kinases will participate in lung oncogenesis (Rikova et al., 2007
) including cMet, which is amplified in some gefitinib-insensitive NSCLC (Engelman et al., 2007
; Lutterbach et al., 2007
). Inspection of the gene expression array dataset derived from the NSCLC cell lines (Coldren et al., 2006
) also predicts the co-expression of the Axl family of receptor tyrosine kinases and their ligands, Gas6 and protein S (Hafizi and Dahlback, 2006
), in a significant fraction of NSCLC cell lines. In fact, a potential role for an Axl/Gas6 autocrine loop in NSCLC has been previously invoked (Shieh et al., 2005
; Wimmel et al., 2001
). Finally, an IGF-1R signaling system has been proposed as a transforming pathway in NSCLC (Morgillo and Lee, 2005
). Our own preliminary studies reveal frequent over-expression of the IGF-1 receptor in NSCLC cell lines, although co-expression of the ligands, IGF1 or IGF2 is less clear. However, it is important to consider paracrine involvement of growth factor receptors as well where the tumor microenvironment may provide growth factors that stimulate receptor tyrosine kinases expressed on lung cancer cells. Combined, our present study and the literature indicate that the EGFR does not function as a single dominant receptor tyrosine kinase in autocrine growth of NSCLC, but that multiple autocrine loops will participate. Thus, effective blockade of autocrine and paracrine signaling in primary NSCLC tumors will require precise identification of the active receptor tyrosine kinase pathways through appropriate biomarkers. The fact that growth factors such as FGF2 are secreted molecules appearing in extracellular fluids of cancer patients (Nguyen et al., 1994
) suggests that the autocrine factors, themselves, may serve as informative biomarkers in this regard.